1. Field of the Invention
The present invention relates generally to the concentration of solutions of macromolecules such as proteins. In one embodiment, the inventive process involves the ultrafiltration of a solution comprising macromolecules and co-concentrating organic polymer, then a diafiltration to reduce conductivity, and then a second ultrafiltration to concentrate the macromolecule without significant loss of yield due to precipitation.
2. Description of Related Art
Protein production involves the creation of large volumes of comparatively dilute protein solution. It is preferred to concentrate the protein after fermentation to facilitate further steps, such as freezing, bulk storage and thawing and downstream purification. However, the ability to achieve optimal protein concentrations for products from solutions containing a co-concentrating organic polymer, such as is found in many cell culture supernatant solutions produced from mammalian cell culture harvest, has proved to be limited in the past due to increased precipitation (and thus loss) of the product molecules as concentration using ultrafiltration increases. For example, excessive precipitation has prevented concentration by ultrafiltration of more than 10-20 fold over the protein concentration in the initial cell culture supernatant.
The isolation, or primary recovery, process forms the interface between fermentation and downstream purification, and is often critical both in terms of production capacity and process economics. The main goal of the isolation process is to obtain the protein product in concentrated, particle-free solution, allowing further processing and downstream purification to be performed.
Ultrafiltration (UF) is an efficient technology for the concentration of protein solutions, and is frequently used as an important step in the isolation of proteins from cell culture supernatant. In particular, ultrafiltration is used in both batch and perfusion cell fermentation processes. Because continuous perfusion processes produce large volumes of comparatively dilute protein product, high concentration factors are desirable to facilitate downstream processes. A major limitation of conventional cell culture based protein manufacturing processes is the attainable concentration factor of protein isolation. In most cases, concentration factors of only 10-20 fold are reached using ultrafiltration during the isolation process. Attempts to increase concentration have resulted in increasing filterability problems and product losses due to precipitation. The nature and cause of the precipitation are generally unknown. Significantly higher concentration in the UF step is typically not easily attainable due to further increased precipitation. This can slow subsequent process steps because very large volumes have to be processed. This is especially true for continuous perfusion processes due to the comparatively low product titer and high volumetric throughput compared to batch fermentation.
Cell culture supernatants consist of a broad spectrum of compounds. These include supplements of the cell culture medium such as nonionic block copolymers, particularly the PLURONIC family of nonionic block copolymers sold by BASE, and silicon oil, and compounds that are secreted from cells or released after cell lysis (e.g., proteins, lipids). The nonionic block copolymer PLURONIC F-68 is usually required as a supplement in cell culture media to protect mammalian cells.
In a preferred embodiment, the present invention is directed to a process to increase the concentration of cell culture supernatant greatly without significant loss of product macromolecule yield or filterability problems. Applicants discovered surprisingly that a cell culture supernatant comprising the product macromolecules and an organic polymer that co-concentrates with the product macromolecules during ultrafiltration, such as a PLURONIC nonionic block copolymer, can be greatly concentrated with higher yields than any reported process of which the Applicants are aware by first subjecting the supernatant to an initial ultrafiltration, then adjusting the conductivity of the retentate, such as by diafiltration with water for injection (WEI), diluent or buffer, and then subjecting the solution to a second ultrafiltration.